Ischemic acute kidney injury (AKI) triggers expression of adaptive (protective) and maladaptive genes. Agents that increase expression of protective genes should provide a therapeutic benefit. We now report that bardoxolone methyl (BARD) ameliorates ischemic murine AKI as assessed by both renal function and pathology. BARD may exert its beneficial effect by increasing expression of genes previously shown to protect against ischemic AKI, NF-E2-related factor 2 (Nrf2), peroxisome proliferator-activated receptor-γ (PPARγ), and heme oxygenase 1 (HO-1). Although we found that BARD alone or ischemia-reperfusion alone increased expression of these genes, the greatest increase occurred after the combination of both ischemia-reperfusion and BARD. BARD had a different mode of action than other agents that regulate PPARγ and Nrf2. Thus we report that BARD regulates PPARγ, not by acting as a ligand but by increasing the amount of PPARγ mRNA and protein. This should increase ligand-independent effects of PPARγ. Similarly, BARD increased Nrf2 mRNA; this increased Nrf2 protein by mechanisms in addition to the prolongation of Nrf2 protein half-life previously reported. Finally, we localized expression of these protective genes after ischemia and BARD treatment. Using double-immunofluorescence staining for CD31 and Nrf2 or PPARγ, we found increased Nrf2 and PPARγ on glomerular endothelia in the cortex; Nrf2 was also present on cortical peritubular capillaries. In contrast, HO-1 was localized to different cells, i.e., tubules and interstitial leukocytes. Although Nrf2-dependent increases in HO-1 have been described, our data suggest that BARD's effects on tubular and leukocyte HO-1 during ischemic AKI may be Nrf2 independent. We also found that BARD ameliorated cisplatin nephrotoxicity.

Ischemic acute kidney injury (AKI) triggers an inflammatory response which exacerbates injury that requires increased expression of endothelial adhesion molecules. To study this further, we used in situ hybridization, immunohistology, and isolated endothelial cells, and found increased Toll-like receptor 4 (TLR4) expression on endothelial cells of the vasa rectae of the inner stripe of the outer medulla of the kidney 4 h after reperfusion. This increase was probably due to reactive oxygen species, known to be generated early during ischemic AKI, because the addition of hydrogen peroxide increased TLR4 expression in MS1 microvascular endothelial cells in vitro. Endothelial TLR4 may regulate adhesion molecule (CD54 and CD62E) expression as they were increased on endothelia of wild-type but not TLR4 knockout mice in vivo. Further, the addition of high-mobility group protein B1, a TLR4 ligand released by injured cells, increased adhesion molecule expression on endothelia isolated from wild-type but not TLR4 knockout mice. TLR4 was localized to proximal tubules in the cortex and outer medulla after 24 h of reperfusion. Thus, at least two different cell types express TLR4, each of which contributes to renal injury by temporally different mechanisms during ischemic AKI.

Although leukocytes infiltrate the kidney during ischemic acute kidney injury (AKI) and release interleukin 6 (IL6), their mechanism of activation is unknown. Here, we tested whether Toll-like receptor 4 (TLR4) on leukocytes mediated this activation by interacting with high-mobility group protein B1 (HMGB1) released by renal cells as a consequence of ischemic kidney injury. We constructed radiation-induced bone marrow chimeras using C3H/HeJ and C57BL/10ScNJ strains of TLR4 (−/−) mice and their respective TLR4 (+/+) wild-type counterparts and studied them at 4 h after an ischemic insult. Leukocytes adopted from TLR4 (+/+) mice infiltrated the kidneys of TLR4 (−/−) mice, and TLR4 (−/−) leukocytes infiltrated the kidneys of TLR4 (+/+) mice but caused little functional renal impairment in each case. Maximal ischemic AKI required both radiosensitive leukocytes and radioresistant renal parenchymal and endothelial cells from TLR4 (+/+) mice. Only TLR4 (−/−) leukocytes produced IL6 in vivo and in response to HMGB1 in vitro. Thus, following infiltration of the injured kidney, leukocytes produce IL6 when their TLR4 receptors interact with HMGB1 released by injured renal cells. This underscores the importance of TLR4 in the pathogenesis of ischemic AKI.

The concepts discussed in this review are important for clinical medicine. Drugs and genetic manipulation may ameliorate ischemic kidney injury by regulating the inflammatory response to cell injury.

ously reported that expression of the transcription factor interferon regulatory factor 1 (IRF1) is an early, critical maladaptive signal expressed by renal tubules during murine ischemic acute kidney injury (AKI). We now show that IRF1 mediates signals from reactive oxygen species (ROS) generated during ischemic AKI and that these signals ultimately result in production of ␣-subtypes of type I interferons (IFN␣s). We found that genetic knockout of the common type I IFN receptor (IFNARI Ϫ/Ϫ ) improved kidney function and histology during AKI. There are major differences in the spatial-temporal production of the two major IFN subtypes, IFN␤ and IFN␣s: IFN␤ expression peaks at 4 h, earlier than IFN␣s, and continues at the same level at 24 h; expression of IFN␣s also increases at 4 h but continues to increase through 24 h. The magnitude of the increase in IFN␣s relative to baseline is much greater than that of IFN␤. We show by immunohistology and study of isolated cells that IFN␤ is produced by renal leukocytes and IFN␣s are produced by renal tubules. IRF1, IFN␣s, and IFNARI were found on the same renal tubules during ischemic AKI. Furthermore, we found that ROS induced IFN␣ expression by renal tubules in vitro. This expression was inhibited by small interfering RNA knockdown of IRF1. Overexpression of IRF1 resulted in the production of IFN␣s. Furthermore, we found that IFN␣ stimulated production of maladaptive proinflammatory CXCL2 by renal tubular cells. Altogether our data support the following autocrine pathway in renal tubular cells:AKI; innate immunity; type I interferon ISCHEMIC ACUTE KIDNEY INJURY (AKI) causes significant shortterm morbidity and mortality (33) and, over the long-term, may contribute to the increasing incidence of end-stage renal disease (15,41,55). Despite these major clinical implications, there is currently no specific therapy beyond supportive care (33). A major insight into pathogenesis was the recognition that the initial ischemic insult elicits maladaptive responses that exacerbate the injury (31). In other words, the ultimate amount of renal injury is determined not only by the direct effects of hypoxia but also by the ensuing maladaptive responses. Understanding the latter may reveal novel therapeutic targets for the treatment of AKI.One maladaptive response is the production of type I interferons (IFNs). Knockout of the gene for the ␣-chain (IFNAR1) of the type I interferon (IFN) receptor heterodimer ameliorated ischemic AKI and decreased renal inflammation in mice (11). Such receptor knockout prevents signaling by, and thus the biological effects of, all type I IFNs (32, 37). These include IFN-, IFN-, IFNε, about which little is known, and also the better understood IFN␤ and the IFN␣ family (which has 13 members in humans, 14 in mice).1 Their biological effects include increasing inflammation and increasing apoptosis (11). These effects should increase injury during ischemic AKI and are in addition to the more widely known antiviral effects of type I IFNs (36, 54).Despite their im...